Chapter 68: Production, Distribution, and Activation of Monocytes and Macrophages

There has been a resurgence of interest in the in situ analysis of macrophages.¹ Genetic/ribonucleic acid interference manipulation, more recently with macrophage-specific/restricted promoters, has been used to knock down macrophage genes or messenger RNA, and to mark cells with fluorescent labels such as green fluorescent protein. Of particular value in tracing their origins and distribution has been the use of fractalkine receptor-transgenics,² and myeloid-specific lysozyme-Cre for targeted ablation.³ Random chemical mutagenesis has been spectacularly successful in validating known, and discovering novel, gene targets that affect macrophage functions.^4,5 A wider range of experimental models (Drosophila, zebra fish) have facilitated interspecies comparisons of macrophage migration and phagocytosis in vivo.^6,7 The analysis of microRNA expression⁸ and functions is still in its infancy and is likely to generate important insights into monocyte/macrophage gene expression in health and disease. Combined with improved imaging methods (fluorescent, nuclear magnetic resonance imaging-based, 2-photon microscopy), new insights have been obtained regarding the dynamic behavior of macrophages and dendritic cells (DCs) in vivo.⁹ There has been progress in provoking embryonic and induced pluripotent stem cell differentiation into macrophages and DCs in vitro, opening the possibility of introducing mutations into human genes, to complement the naturally occurring material derived from human inborn errors and resultant genetic diseases.¹⁰

Although individual-labeled cells can be followed in accessible tissues or ex vivo, the resolution, isolation, and characterization of important embedded macrophage populations are limiting. Methods of isolation from solid organs, for example, brain and even liver and gut, are prone to artifact, and macrophages are profoundly affected by removal from their natural tissue environment. Many of the genetic manipulations introduced by transgenesis are leaky and not uniform, not surprising in the light of macrophage heterogeneity. Although the fate of recently recruited cells from blood into tissues can be tracked more easily, the slowly turning over resident populations are less easily accessed, resulting in bias. Finally, there are intrinsic difficulties with human experimentation in vivo. Induced skin blisters, for example, make it possible to collect fluid and cells from sites of inflammation.¹¹ However, the low frequency of monocytes compared with neutrophils limits the use of ex vivo indium-labeled cells for transfer studies in vivo.

DEVELOPMENT OF MONOCYTES AND MACROPHAGES

Macrophages and related amoeboid phagocytic cells, ancient in the evolution of multicellular organisms, are the main leukocytes responsible for innate immunity and tissue remodeling, as documented by Metchnikoff in his pioneering studies on invertebrates,¹² and confirmed by contemporary studies on Drosophila melanogaster.⁷ In mammals, much of our knowledge of macrophage ontogeny derives from studies in the mouse. After origins from an aortic mesonephric site, the best understood phases of macrophage development occur during midfetal development, in the yolk sac, followed by fetal liver, spleen, and marrow, before and after birth.¹³ The association of macrophages with definitive erythropoiesis is a striking feature of fetal liver hematopoiesis from approximately day 12 of mouse development; macrophages then, for the first time, become intimately associated with nucleated erythroblasts, reaching a peak of hematopoietic cluster formation at day 14. The role of stromal macrophages in hematopoiesis within the adult is illustrated and discussed further in this chapter.

The association of macrophages with erythroblasts is mediated by surface adhesion molecules,¹⁴ including a poorly characterized divalent cation-dependent receptor and the sialic acid-binding molecule sialoadhesin (Siglec1).¹⁵ The potential trophic functions of stromal macrophages in marrow erythropoietic islands is poorly understood, as is the considerable role of macrophages in iron and heme metabolism. Macrophages interact with cells in numerous ways; however, during erythropoiesis a special phagocytic process allows for the removal of pyknotic erythroid nuclei during the final stages of erythropoiesis. The mechanism of recognition of membrane-bound erythroid nuclei is not clear, nor its relationship to the uptake of apoptotic cells elsewhere during development. The production of granulocytes from progenitors also involves macrophage–myeloblast clusters and similar adhesion receptors. Once fetal liver hematopoiesis declines before and after birth, the macrophages in the liver adopt the features of resident Kupffer cells. The stromal macrophages associate with developing blood cells within islands of clustered cells, a feature of hematopoiesis throughout life.¹⁶ During fetal life, monocytes and macrophages are distributed through the developing vasculature, providing amoeboid, phagocytic cells implicated in tissue remodeling, for example, sculpting of digits,¹⁷ and growth of the central nervous system.¹⁸ Blood monocytes seed resident tissue macrophage populations throughout the organism, and these cells proliferate more readily in the fetus than in later life; the adhesion molecules, chemotactic signals, and receptors involved during this constitutive phase of distribution are poorly defined, but it is independent of the β₂-integrin CD11b/CD18, which plays a role in myelomonocytic cell recruitment induced by inflammation in the adult.¹⁹ The appearance of macrophages during development has been correlated with fibrous scar formation after injury.¹⁷ In sum, macrophages play a major role during development, both in hematopoiesis and in extravascular tissues, and much remains to be learned regarding their properties in the fetus.

Growth, Differentiation, and Turnover

Figure 68–1 gives an overview of differentiation of monocytic cells in the adult.²⁰ The origins of monocytes from multipotential (progenitors colony-forming units, spleen [CFU-S]) and committed hematopoietic precursors (colony-forming units, culture [CFU-C]) and the role of lineage-restricted growth factors such as monocyte/macrophage colony-stimulating factor (M-CSF; also termed CSF-1) and granulocyte-macrophage (GM)-CSF have been studied extensively, but new details are still emerging. Both transcription factor c-Myb and receptor FLT3, M-CSF–dependent myeloid lineages (monocyte and dendritic) occur. These cell types may have different responses to tissue damage and infection.²⁰ Monocytes share precursors with other hematopoietic cells and are closely related to granulocytes. Monocytic precursors are the source of adult tissue macrophages, as well as of myeloid DCs and osteoclasts. Their relationship to B lymphocytes and to plasmacytoid DCs is still unclear, as plasmacytoid DCs express a range of myeloid as well as lymphoid markers. There is a considerable body of knowledge about the specific growth factors and their receptors, and growing knowledge of the nature and role of transcription factors involved in monocyte/macrophage differentiation.²¹ Genetic and cellular abnormalities in growth and differentiation pathways underlie myeloid leukemogenesis, though rarely giving rise to monocytic leukemia.

MATURATION AND DIFFERENTIATION OF MONOCYTES AND MACROPHAGES

The classic studies of Lewis and Lewis²² in 1926, Maximow²³ in 1932, and Ebert and Florey²⁴ in 1939, showed that monocytes transform into macrophages and multinucleated giant cells in vitro. Macrophages can be produced from monocytes or hematopoietic progenitor cells culture in cytokines, such as GM-CSF or M-CSF.

The alterations of ultrastructure during transformation into macrophages, epithelioid cells, and giant cells have been described using purified populations of monocytes and in vitro culture techniques.²⁵ As the monocyte matures into the macrophage, the cell enlarges in size, and the lysosomal content and the amount of hydrolytic enzymes within the lysosomes (e.g., phosphatases, esterases, β-glucuronidase, lysozyme, arylsulfatase) increase. At the time, the size and number of mitochondria increase, their energy metabolism increases concomitantly. Production of lactate also increases. The Golgi complex, which packages lysosomes, increases in size and vesicle complexity (Chap. 67). Several stimuli induce formation of multinucleated giant cells from monocytes.²⁶

Growth Factors

M-CSF and GM-CSF are the major growth factors implicated in monocyte and macrophage differentiation. Other cytokines, such as interleukin (IL)-3 and IL-4, result in minimal monocyte proliferative expansion, and their genetic elimination has no effect on the lineage. M-CSF promotes survival as well as growth and differentiation of macrophages, exclusively, acting through a specific receptor (CSF-1R), encoded by the protooncogene c-FMS, which has been extensively used as a lineage marker for fluorescence-activated cell sorting (FACS) analysis (CD115) and transgenesis.^27,28 The role of M-CSF has been reviewed²⁹ and its role in macrophage and osteoclast development is illustrated in Fig. 68–2. The naturally occurring mouse mutant, op/op, gives rise to M-CSF deficiency and osteopetrosis, with marked or partial deficiency in monocyte and selected tissue macrophage populations; DC numbers are unaffected.³⁰ Unlike PU.1 deficiency, the op/op mouse is viable, though its reproductive ability is impaired, because M-CSF also plays an important role in the reproductive system. Uterine epithelium is a rich source of M-CSF, inducing monocyte-macrophage recruitment, growth and differentiation, and upregulating scavenger receptor (SR) expression, cell adhesion, and endocytosis of modified low-density lipoproteins and other polyanionic ligands. M-CSF is produced in a soluble and membrane-bound forms, is present in plasma, and has been implicated in atherosclerosis and tumor-dependent recruitment of monocytes and macrophages. The size of the growth burst induced by M-CSF depends on the stage of differentiation of the target cell, decreasing markedly as the precursors mature into monocytes and macrophages. Adhesion and inflammatory stimuli enhance the response to growth factors and can result in macrophage proliferation at peripheral sites, for example, in granulomata.

GM-CSF has a broader myeloid target profile. It is produced by many cells, including macrophages themselves, especially after inflammatory stimuli such as lipopolysaccharide (LPS), and it enhances production of monocytes and macrophages with a different morphology to that induced by M-CSF. GM-CSF is required for myeloid DC differentiation in vitro and has been widely used, alone and in combination with cytokines such as IL-4 or transforming growth factor (TGF)-β, to produce DC from mouse marrow or from human monocytes in cell culture.^31,32 Its targeted deletion in mice or genetic loss of function mutants of its specific receptor chain in humans results in pulmonary alveolar proteinosis, associated with defective alveolar macrophage metabolism of pulmonary surfactant.³³

Survival, Differentiation, and Turnover Overview

Once the cells have acquired the characteristics of mature monocytes/macrophages, they display considerable heterogeneity in morphology and phenotypic plasticity. In general, their proliferative potential is limited, and their life span can vary from less than 1 day to many months, depending on their microenvironment, infections, and other stimuli. Although terminally differentiated, macrophages remain extremely active in messenger RNA and protein synthesis, with complex, often characteristic profiles of gene expression, depending on innate and acquired immune stimuli and cellular interactions. Tissue macrophages are relatively resistant to apoptosis, compared with neutrophils, but this feature changes during infection. Their active membrane turnover and endocytosis make them susceptible to toxic agents, making them targets for clearance by surviving macrophages. Sublethal injury and infection can also induce autophagy, increasingly recognized as an important component of inflammatory and infectious diseases.

The remarkable ability of macrophages to undergo homotypic cell–cell fusion results in giant cell formation. This is a feature of osteoclast differentiation, depending on M-CSF and the tumor necrosis factor (TNF) family member receptor activator of nuclear factor-κB ligand (RANKL), which act on monocytic precursors to yield catabolic cells able to excavate and remodel living bone. Local adhesion and ruffling of their plasma membrane are associated with focal, polarized release of H⁺ and hydrolytic enzymes by monocyte-derived osteoclasts. The attempted uptake of non- or poorly degradable foreign materials induces “foreign-body giant cells,” with distinct properties; macrophage-derived giant cells are also characteristic of granulomatous diseases such as tuberculosis (Langerhans giant cells; Fig. 68–3) and parasitic infections (e.g., schistosomiasis). Mycobacterial and ill-defined host lipids are able to induce giant cell formation in vitro. The mechanism of fusion involves cellular differentiation to induce a fusogenic phenotype and surface glycoprotein interactions with a selected substratum; T-helper type 2 (Th2) cytokines, such as IL-4 and IL-13, act through a common receptor chain and signaling pathway to enhance macrophage fusion.³⁴ DNA synthesis, a feature of high-turnover granulomata associated with infection, can result in abortive cell division and cell death. These macrophage-derived giant cells are distinct from syncytia induced by fusogenic virus infection, especially paramyxoviruses and retroviruses such as the human immunodeficiency virus.

HETEROGENEITY

Monocytes are defined as the population of differentiated cells present in the circulation, with classical morphologic features (Chap. 67), and include the less-well defined precursors able to give rise to myeloid DCs and osteoclasts. Because of their ready availability from human blood and the sensitive methods now available to analyze their phenotype ex vivo (FACS, microarray, immunochemistry, and cytochemistry), human monocytes have been more amenable to study, whereas in the mouse, analysis of precursor–product relationship and tissue distribution have provided new insights into the fate and heterogeneity of the circulating population. The number of monocytes in the circulation depends on constitutive, steady-state production and delivery from marrow, possibly from marginated pools in spleen, as well as adhesion and diapedesis in response to unknown stimuli and enhanced recruitment in response to peripheral stimuli such as infection and inflammation. M-CSF and glucocorticoids affect their level and phenotype, as do metabolic stimuli; Chap. 70 describes clinical conditions that give rise to monocytosis. The biochemical properties and functions of monocytes are described in Chap. 67. They are relatively radioresistant once entering the circulation, where they persist for 12 to 48 hours as motile cells, with an ability to engulf particles and to adhere transiently or more stably to arterial as well as microvascular endothelium, thus modulating their phagocytic ability. Depending on interactions with the vessel wall and local differentiation, monocytes are able to crawl along and patrol the intravascular surface utilizing CD11a, a β₂-integrin–dependent property.²⁰ Mature macrophages lining the endothelium can also detach and recirculate, for example, filled with lipid stores as foam cells in atherosclerosis, and circulate heavily laden with erythroid breakdown products in malaria.

The presence of significant numbers of immune cells and molecules in adipose tissue suggest vibrant interactions between the immune and metabolic systems. In obesity, the inflammatory infiltrate and activation state of macrophages in adipose tissue may contribute to insulin resistance. The cellular localization and inflammatory potentials of macrophages,³⁵ as well as the ratio of macrophages to adipocytes,³⁶ differ in obese and lean mice. In lean mice, macrophages in the adipose tissue have the alternate or M2 phenotype (ARG1+CD206+CD301+), are uniformly distributed, and serve a protective function as they are less inflammatory and promote insulin sensitivity by producing IL-10; however, in obese mice, macrophages distribute around necrotic adipocytes, and induce inflammation and insulin resistance.^35,37 CC-chemokine receptor 2 (CCR2) and its ligand (CCL2) are critical for macrophage recruitment to adipose tissue.³⁸ Metabolic disease can be viewed as maladaptive consequence of inflammation-induced insulin resistance, which may beneficially conserve energy resources for the immune system combatting infection for brief periods.^{39,40,41,42,43}

The precursors of myeloid DC and osteoclasts may represent a subpopulation of monocytes, whose further differentiation depends on cytokines and local factors in the vessel wall, marrow, and other tissues. Ex vivo substantial numbers of monocytes give rise to myeloid DC after treatment with GM-CSF and IL-4.³² Monocytes that differentiate into macrophages do not recirculate for the most part, but persist for varying times as “resident” tissue cells that turn over locally, especially in lymph nodes. It is not known if the constitutive exit from blood is a stochastic process or specific to particular tissues.

Phenotypic heterogeneity of monocyte populations has become a topic of intense interest, thanks to the availability of surface antigens/receptors such as CD14, CD16 (human), and Ly6C (mouse), and analysis of chemokine/receptor expression, especially fractalkine receptor (CX₃CR) and CCR2.³⁴ Figure 68–4 illustrates the subsets and tissue progeny established by the use of genetically manipulated mice and Table 68–1 compares expression of markers to characterize monocyte subsets in mouse and human blood. The relationship of monocyte precursor subsets that give rise to inflammatory tissue macrophages and DCs is better defined than is that of those that give rise to resident cells, which turn over more slowly. Current studies aim to elucidate the subset origin of other recruited populations, for example, in atherosclerosis, normal CNS, and tumors, and in response to metabolic, traumatic, or degenerative injury. Conceptually, it is still not clear how stable these apparently distinct subsets are or whether they represent part of a continuous phenotypic spectrum, arising by modulation of subpopulations rather than irreversible, true differentiation. Separation and microarray analysis of freshly isolated monocytes will yield further information regarding this question, providing novel markers and diagnostic signatures. Removal from an in vivo environment, as well as in vitro artifacts, can profoundly alter the phenotype and function of monocytes in such studies. Imaging and in situ analysis may enable single-cell direct studies of their fate.

Monocyte/macrophages have a major role in the development and progression of cardiovascular disease.^44,45 In acute myocardial infarction macrophages with an M1 proinflammatory profile migrate to the cardiac tissue and are involved in cardiac remodeling (Chap. 134).⁴⁶

In atherogenesis, there is recruitment of monocytes into the vascular wall at sites of turbulent flow. Once within the subendothelial tissue, the monocytes differentiate into macrophages and engulf oxidized low-density lipoprotein accumulated in arteries, leading to foam cell formation, atheroma development, and the secretion of profibrotic agents by adjacent vascular smooth muscle cells, resulting in a fibrous cap formation. Thus, vascular wall macrophages are key factors in initiating the atherosclerotic lesion. Moreover, macrophages activate the coagulation cascade (Chap. 67) inducing thrombus formation and vascular occlusion.

Resident Macrophage Populations in Adult Tissues Overview

It is important to describe first the nature of those macrophages present throughout the body as resident populations, in the absence of overt inflammation, before considering the altered monocyte-derived macrophages recruited to local sites by infectious or sterile inflammatory (e.g., metabolic) stimuli. The properties of such elicited macrophages are well established and are described in Chap. 67. However the functions of resident macrophages, especially in different organs, are still mysterious and are considered in outline here, with further details in Chap. 67.

The use of differentiation antigens such as F4/80 and cd68 (mouse) and CD68 (human) has made it possible to define resident macrophage populations in mouse tissues,⁴⁷ and to compare their anatomic relationships in the two species (Table 68–2). F4/80 (EMR1), a member of a family of epidermal growth factor-7 transmembrane (EGF-TM7) plasma membrane molecules, is broadly present and almost exclusive to macrophages (Fig. 68–5A to C).^48,49 It is related to G-protein–coupled chemokine receptors in structure, but has a large epidermal growth factor (EGF) domain extracellular extension, thought to be involved in adhesion to extracellular matrix. The human members of this family are more broadly present on myeloid cells; EGF module-containing mucin-like hormone receptor 2 (EMR2) is a useful tissue marker for human macrophages, although it is also present in neutrophils and immature DCs (Fig. 68–5A). Additional macrophage antigen markers useful for immunocytochemical and FACS analysis include Siglec1 (Fig. 68–5D), a sialic acid-binding lectin, the β₂ integrins CD11b/CD18 (Mac1, CR3), and CD11c, present on DCs and selected, especially alveolar, macrophages.⁵⁰ Receptor antigen markers include SR-A,⁵¹ a broadly expressed macrophage receptor additionally found on sinusoidal endothelium, whereas MARCO (macrophage receptor with collagenous structure), a related collagenous SR, is more restricted in expression.⁵² Additional markers include lectins such as the macrophage mannose/fucose receptor (MR; Fig. 68–5E).⁵³ CD163, a receptor for hemoglobin–haptoglobin complexes, is induced by glucocorticoids,⁵⁴ IL-10,⁵⁴ and substance P.⁵⁵ Complement receptors (CRs) and Fc receptors (FcRs) are described in Chap. 67.

The resident macrophages in tissues constitute a major dispersed organ system, responsive to endogenous and exogenous stimuli; they are highly active in uptake of particles and soluble ligands, providing not only sentinels for defense at portals of entry, but also mediating the clearance of damaged or dying cells and modulating the properties of viable neighboring cells. In sum, these cells provide a homeostatic, trophic function that is often overlooked in considering their role in cytotoxicity and antimicrobial host defense. The properties of macrophages in hematolymphoid organs and other tissues, with special relevance to hematologic aspects, are discussed in detail.

HEMATOPOIETIC ORGANS

Marrow

It is often overlooked that mature macrophages are important constituents of the hematopoietic stroma,⁵⁶ along with fibroblastic mesenchymal cells, osteoblasts, and endothelial cells, contributing to hematopoiesis beyond their own differentiation (Figs. 68–6A to E and 68–7). Stromal macrophages in hematopoietic island clusters associate with developing erythroid and other granulocytic cells through nonphagocytic, cell–cell adhesion receptors, such as sialoadhesin and a divalent cation-dependent receptor, as described for fetal liver. The potential trophic functions provided by stromal macrophages are ill-defined but include surface-expressed and secreted growth factors and cytokines. Stromal macrophages are actively endocytic and clear erythroid nuclei and apoptotic hematopoietic cells as required, rapidly degrading them for possible reutilization of iron and other nutrients. Stromal macrophages also interact with less-differentiated hematopoietic precursors through release of potent secretory products, such as IL-1, and with lymphocytic populations, including plasma cells, through IL-6. They are targets for infectious agents, for example, mycobacteria, lentiviruses, and retroviruses, and serve as reservoirs in many chronic infections, while expressing a reduced killing capacity, as demonstrated for other resident macrophage populations.

Several monocyte and macrophage populations coexist in the marrow compartment; a network of stromal macrophages, clustered in hematopoietic islands, the developing monocytes, as well as osteoclasts and isolated macrophages in apposition to bone surfaces. Mature macrophages in human marrow contain prominent inclusions in storage disorders, such as Gaucher disease and hemosiderosis. Hemophagocytosis, a consequence of perforin deficiency in some patients, and seen in genetic syndromes and postviral infection, is a striking manifestation of excessive macrophage cytopathic activity in the marrow.^57,58 Uptake of opsonized platelets by macrophage FcR and CRs in stromal and other resident tissue macrophages are important features of thrombocytopenic syndromes.

The hematopoietic stem cell lineage which gives rise to monocyte-macrophages and myeloid DCs also leads to production of the osteoclast lineage.^59,60 Following interaction via Stat4 and RANKL (regulator of activation of nuclear factor-κB), a member of the superfamily of TNF, cells undergo differentiation, fusion, attachment to bone as osteoclasts and then function in bone remodeling.⁶¹

A common marrow progenitor cell that gives rise to both monocytes and DCs has been defined,²⁰ including both classical DCs and the plasmacytoid DCs.^62,63 This common marrow progenitor cell circulates in the blood and seeds lymphatic tissues.^62,63 These short-lived, migratory cells modify T-cell responses and, unlike Langerhans cells, are replaced by bloodborne precursors.^41,42 The central activity of immature classical DCs is phagocytosis, while that of mature classical DCs is cytokine production.^62,63

Dendritic cells that occur in lymphoid and nonlymphoid organs have a major role in processing and presenting antigens, leading to unique T cell function. The “classical” DC develops from a precursor which is dependent on the growth factor receptor.^62,63,64

Spleen

From the macrophage point of view, the spleen is the most complex organ in the body.^65,66 Our knowledge is based mainly on the mouse and we know there is considerable species variation,⁶⁷ as well as constitutive hematopoiesis in mouse spleen. Subpopulations observed in the mouse by marker and genetic knockout experiments include (1) macrophages in the red pulp, white pulp, and in the marginal zone, itself M-CSF dependent,³⁰ and (2) heterogeneous, more phagocytic “metallophilic” macrophages in the outer marginal zone. Characteristic phenotypic markers are available to identify macrophages in mouse spleen (see Fig. 68–5C to E). The F4/80 antigen and the mannose receptor (MR) are restricted to mature macrophages in the red pulp, whereas CD68 is a marker for all macrophages, as well as DCs, although the mainly intracellular expression of CD68 is less prominent in DCs. There are several well-characterized markers for mouse metallophilic macrophages, including sialoadhesin (Sn), a poorly characterized protein recognized by the MOMA-1 monoclonal antibody, and ligands for MR cysteine-rich domain-Fc chimeric proteins (see Fig. 68–5E). The splenic marginal zone macrophage population develops postnatally,⁶⁸ in parallel with antipolysaccharide responses to encapsulated bacteria. Functions of splenic marginal zone macrophages include clearance of senescent erythrocytes and neutrophils (red pulp), targeting of circulating antigens and pathogens (marginal zone), interferon (IFN) production, induction of secondary adaptive immune responses, regulation of hematopoiesis, and iron storage. Markers for the outer marginal zone macrophages include MARCO and SIGNR1, a mouse homologue of dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN). The spleen is also a site for storage and rapid deployment of monocytes, which participate in wound healing and play a role regulation of inflammation.⁶⁹

Lymph Nodes

Lymph node macrophages are also heterogeneous, with distinctive Sn subcapsular cells, corresponding to marginal metallophils in their marker expression, and F4/80+ macrophages in germinal follicles and in the hilus. Macrophages in T-lymphocyte–rich areas are F4/80– or dim, as in T-cell areas in spleen, but express CD68. It is thought that antigen enters lymph nodes via afferent lymphatics and two photon experiments have defined the possible transfer of viral and other antigens and immune complexes to B lymphocytes after capture by the subcapsular sinus macrophages.⁷⁰ Their contributions to the initiation of adaptive immune responses, compared with DCs, are unclear. Tingible body macrophages arise from the clearance of apoptotic B cells in germinal centers, as in the spleen.

Nonlymphohematopoietic Organs

In bulk, the gastrointestinal tract represents the largest accumulation of F4/80+ macrophages in the body, extending throughout the upper and lower gut. The small intestine is essentially sterile, and the abundant F4/80+ resident macrophages in the lamina propria express a distinct phenotype, ascribed to TGF-β production by adjacent cells.^56,71,72,73 The liver contains an abundant population of sinusoidal F4/80+ Kupffer cells, which share some properties (FcR, MR, SR-A) with sinusoidal endothelium, which lacks F4/80. The skin has F4/80+ epidermal Langerhans cells and F4/80+ dermal macrophages, which can migrate to draining lymph nodes and differentiate into antigen-presenting DCs.^74,75 The lung has a distinctive F4/80– or dim alveolar macrophage population, as well as interstitial F4/80+ macrophages. Alveolar macrophages are CD11c+ and express a range of nonopsonic phagocytic receptors (MR, SR-A), as well as FcR, but lack CR3. These cells also contain particle debris, cigarette smoke residue, and abundant lysozyme, because of exposure to irritants and uptake of carbon and dust particles, as well as of mucosal secretions in the airway.

The central nervous system contains an extensive network of F4/80+ CR3+ microglia, derived from monocytes during development, when they remove apoptotic neurons.¹⁸ They differentiate into characteristic membrane-rich arborized forms within the neuropil and persist throughout adult life. Their function is obscure but may involve homeostasis and catabolism of neurotransmitters. In addition, there are perivascular F4/80+ macrophages (also MR+ SR-A+) and other F4/80+ populations in the meningeal space and choroid plexus. The endocrine, exocrine, reproductive, and urinary tracts all contain macrophage populations at sites of phagocytosis (ovary, testes) and hormonal metabolism (adrenal, thyroid, for example).⁷¹ Precise characterization of these cell types using monoclonal antibodies with specificity for human cell types in tissue requires further analysis.

RECRUITMENT OF MONOCYTES IN RESPONSE TO INFLAMMATION AND TUMORS

The stimuli that give rise to induced recruitment of monocytes, with or without accompanying myeloid and/or lymphoid cells, and the mechanisms involved are better understood than those of constitutive tissue localization. Bacterial infections induce enhanced myelomonocytic cell recruitment and follow the stages established for neutrophils, transient arrest, and rolling on the microvascular endothelium, mediated by L-selectin, and initiated by chemotactic stimuli acting via G-protein-coupled chemokine receptors (Fig. 68–8). The β₂ integrins CD11a/CD18 and CD11b/CD18 mediate more stable adhesion. This is followed by diapedesis and interactions with CD31. Receptors implicated in subsequent extravascular migration are less defined but may include the fractalkine receptor, and intravascularly, β₁- and β₂-integrin, CD44, and EMR2. The evidence for an important role of L-selectin and β₂ integrins in human phagocyte recruitment to inflammatory stimuli comes from human inborn error syndromes, mouse genetic experiments, and antibody inhibition. The role of the common β₂-integrin chain (CD18) and definition of leukocyte adhesion deficiency syndrome provided a powerful paradigm for further experimental study of CD11/CD18.^76,77 Cellular signaling gives rise to dynamic changes in migration/adhesion and cytoskeletal reorganization outlined further in Chap. 67. Studies of tumor-associated macrophages (TAMs) in mouse model systems, particularly mammary cancer, have identified a population of unique macrophages. The TAMs develop from marrow-derived macrophages that are inflammatory phenotypes and are recruited to the tumor.^78,79

Mononuclear cell recruitment without that of other myeloid cells is a feature of viral infection and modified forms of inflammation observed in metabolic diseases, atherosclerosis, storage disorders, autoimmunity, and tumors. Different chemokine receptors and cell adhesion molecules account, in part, for more selective monocytic recruitment, although some are shared. The phenotypic heterogeneity in monocyte subsets is characterized by quantitative differences in expression of plasma membrane molecules resulting in differential recruitment of subsets in response to different stimuli. Once in the tissues, their subsequent fate also varies markedly, depending on the local environment, where newly recruited monocytes respond to tissue-specific factors. A striking example is that observed in the neutrophil, where monocytes can differentiate over a few days into highly arborized, activated microglia, resembling locally reactivated resident microglia.¹⁸ Thus, it becomes progressively more difficult to distinguish newly recruited from initially resident cells through marker analysis. Direct observation by fluorescent imaging in vivo may define precursor–product relationships more clearly. Similar issues arise in other organs, for example, lung, liver, gut, and even in skin, where static observations can be misleading. There are also common features of recruited cells irrespective of the local tissue environment, including the expression of CD11b/CD18 and monocytic adhesion molecules, and metabolic markers, such as the ability to undergo a respiratory burst and an increased proliferative potential and high cell turnover rate. These monocytic markers tend to decline upon further macrophage differentiation, and in the case of myeloperoxidase may not be renewed after degranulation.

HETEROGENEITY OF MACROPHAGES IN TISSUES: IMMUNOMODULATION

Characterization of the macrophages found in tissues has yielded insights into their versatility in response to microbial constituents and cytokines produced by lymphoid, other immune and nonimmune cells. Adhesion to extracellular matrix, metabolites, vascular, and hormonal changes all influence the macrophage phenotype. This variety of stimuli can selectively activate or deactivate macrophage gene and protein expression, regulating their function. Figure 68–9 illustrates some of the stereotypic signature phenotypes, and Chap. 67 further describes the innate recognition mechanisms and functional responses.

Broadly considered, it is convenient to distinguish several clusters of activation properties; innate, classical, and alternative activation, and deactivation. The definition of activation has a long and confusing history, has mainly been based on limited models of analysis, typically peritoneal macrophages in vivo and in vitro, and on studies with macrophage-like cell lines. The advent of microarrays and of proteomics and systems biology has generated increasingly detailed information. It makes sense to schematize the interactions of monocytes and macrophages with microorganisms, microbial products, and Th1/Th2 lymphocytes, although this is subject to revision as CD4 T-lymphocyte heterogeneity continues to grow in complexity, with the description of Th17, FoxP3+, and other regulatory T cells. Further details are given in Chap. 67.

INNATE ACTIVATION

For this discussion, innate activation is defined as direct microbial stimulation by intact bacteria or their constituents, such as LPS acting via toll-like receptor (TLR) sensors, in the absence of the major Th1/2 cytokines. For example, ethanol-killed Neisseria meningitidis, a potent immunomodulator with adjuvant-like properties, stimulates the expression of two useful markers on macrophages: MARCO and CD200. Expression of MARCO, a class A SR, is remarkably specific for macrophages (and DCs) and is regulated developmentally on the outer marginal zone macrophages, but it is inducible on most macrophage populations by TLR and myeloid differentiation factor 88 (MyD88)-dependent bacterial stimuli. It is a phagocytic and adhesion receptor providing an adaptive, enhanced ability to take up Neisseria and other bacteria, after innate activation. CD200, an immunoglobulin (Ig) superfamily member, is widely expressed on many cells, but not on resident macrophages, and part of an immunoregulatory receptor pair with CD200 R, is also induced on macrophages by innate stimuli.

Lectins, such as Dectin-1, control innate activation of macrophages by β glucans in fungal walls,⁸⁰ in collaboration with TLR pathways, as discussed in Chap. 67. TLR-independent innate activation by viruses, parasites, and other pathogen-associated stimuli requires further study.

CYTOKINE-INDUCED PRIMING AND ACTIVATION: CLASSICAL AND ALTERNATIVE ACTIVATION

Investigations in normal humans, pathologic states, and animal models have led to the characterization of different states of macrophage polarization (Fig. 68–10).⁸¹ The terms macrophage “activation and “polarization” require well-defined nomenclature and experimental guidelines. It has been recommended that the term “activation” be used to refer to perturbation of macrophages with exogenous agents the same way that many authors use “polarization.”⁸² Studies of in vitro, ex vivo, and in vivo cells from animals and humans have led to descriptions of complex cellular structural, biochemical, and functional activation states of macrophages. Consequently, it is essential to describe the properties of the macrophage its activation/polarization state. There are various nomenclatures and Fig. 68–10 indicates the major features of the M1–M2 dichotomy. This dichotomy, however, is more of a continuum and there are transitional states. The most useful approach is to describe the spectrum of activation states by cell isolation technique, membrane receptors, cytokines, chemokines, and metabolic markers, and when possible, genetic modifications producing shifts in activation phenotype. This approach is essential for deciphering the role of monocyte–macrophages in disease pathogenesis.

Macrophage polarization usually is driven by unique pattern recognition receptors (PRRs). Classical activation of macrophages or M1 in general⁸³ have a proinflammatory profile. This pathway is triggered by IFN-γ followed by a microbial stimulus, LPS. The M1 macrophage is characterized by high antigen presentation and production of IL-12, IL-23, nitric oxide, and proinflammatory cytokines, including IL-1, TNF-α, IL-6, and CXCL-1, -2, -3, -5, -8, -9, and -10. The pathway to the alternative M2 macrophage is mediated by IL-4, IL-10, and IL-13. These cells demonstrate enhanced expression of Dectin-1, MR C1 (CD206), CD163, CCR2, CXCR1, CXCR2, and DC-SIGN.⁸⁴ M2 produce high levels of IL-10 and low-levels of IL-12.⁸⁵ IFN-γ, produced mainly by natural killer cells and activated Th1 CD4+ and CD8+ cytotoxic lymphocytes, induces a set of macrophage biosynthetic and effector responses, known as classical activation, because of its well-established role in enhanced macrophage functions in cell-mediated immunity, inflammation, and host defense, particularly against intracellular pathogens. Full activation of effector functions, such as the respiratory burst and generation of oxidative nitrogen metabolites, depends on a two-stage mechanism of priming by the cytokine, via specific IFN-γ receptors, followed by a local stimulus, LPS, or other TLR ligands. Although essential for host defense, including against opportunistic pathogens such as found in patients with the acquired immunodeficiency syndrome, classical activation is responsible for tissue injury and its consequences in inflammatory bowel disease, tuberculosis, and rheumatoid arthritis, although additional immunopathogenic agents, such as immune complexes, also contribute. Biochemical and cellular aspects of classical activation are described in Chap. 67.

The Th2 cytokines IL-4 and IL-13, acting via a common receptor chain as well as distinct receptors, induce a characteristic signature of altered gene expression in macrophages known as alternative activation.^86,87 Such primed macrophages can be induced to respond further to local, TLR-dependent phagocytic stimuli to secrete enhanced levels of proinflammatory cytokines, analogous to classically activated macrophages. Alternative activation is associated with allergy and parasitic infection, and has been implicated in humoral immunity, control of Th1-dependent inflammation, and host defense to extracellular pathogens, such as helminths. It can promote repair or, if excessive, fibrosis. Other forms of alternative activation have been described after stimulation of macrophages by immune complexes, acting via FcR. A major G-protein–coupled receptor that is important in macrophage activation is the Neurokinin-1 receptor.⁸⁸ This receptor, which has a full-length form and a truncated splice variant, is important in macrophage signaling and calcium fluxes.^89,90 An important caveat is that there are substantial species differences in the marker changes depending on the differentiation and prior activation state of the macrophages.

IL-10 is a major deactivating cytokine for macrophages, produced by the macrophages themselves, as well as by Th2 lymphocytes and other sources. Acting through its own receptor, it counteracts IFN-γ, and can potentiate IL-4–induced actions. Other antiinflammatory regulators of macrophages activation include glucocorticoids and prostaglandin E₂. Although less-well defined, the overall gene and protein expression profiles of macrophages are also markedly influenced by the extracellular matrix, hormones, and other immunomodulators, so that modified forms of inflammation are associated with macrophages present in lipid-rich environments, tumors, and metabolic diseases. Finally, cell–cell interactions, as well as intracellular regulatory networks, profoundly influence the functions of macrophages, and are described in Chap. 67.